Feasible low bone density condition for assessing bioactivity in ex-in vivo and in vivo studies

Abstract Objective To choose a critical animal model for assessments of bone repair with implant installation by comparing senile rats (SENIL) to young ovariectomized rats (OXV). Methodology For the ex-in vivo study, the femurs were precursors for bone marrow mesenchymal stem cells. Cellular responses were performed, including cell viability, gene expression of osteoblastic markers, bone sialoprotein immunolocalization, alkaline phosphatase activity, and mineralized matrix formation. For the in vivo study, the animals received implants in the region of the bilateral tibial metaphysis for histometric, microtomography, reverse torque, and confocal microscopy. Results Cell viability showed that the SENIL group had lower growth than OVX. Gene expression showed more critical responses for the SENIL group (p<0.05). The alkaline phosphatase activity obtained a lower expression in the SENIL group, as for the mineralization nodules (p<0.05). The in vivo histological parameters and biomechanical analysis showed lower data for the SENIL group. The confocal microscopy indicated the presence of a fragile bone in the SENIL group. The microtomography was similar between the groups. The histometry of the SENIL group showed the lowest values (p<0.05). Conclusion In experimental studies with assessments of bone repair using implant installation, the senile model promotes the most critical bone condition, allowing a better investigation of the properties of biomaterials and topographic changes.


Introduction
The bone tissue undergoes changes in its microarchitecture life-long, physiologically due to the impact of body weight, being more expressive in women after menopause due to estrogen depletion.¹ Thus, molecular damage adds up to a drastic drop in tissue-specific progenitor cells, and there is a functional decline in tissue homeostasis, which may lead to osteopenia or osteoporosis,² defined as "a systemic skeletal disease characterized by low bone mass and complaint in the microarchitecture of bone tissue, with consequent increase of the bone fragility and susceptibility to fracture."³ The incidence of osteoporosis has increased exponentially, reaching an average of 150 million people worldwide. 1 Therefore, it is important to choose an osteopenic animal model to evaluate new treatments, prevention, or intervention for osteopenia.
Moreover, it is an FDA requirement that these therapies have their efficacy proven in a small and large animal models with known intracortical bone remodeling. 4 Today, studies that evaluate the properties, quality, and metabolic responses of the most diverse biomaterials have been using the osteoporotic animal study model due to the possibility of evaluating the adaptation, regeneration, stability, preservation, duration, and bioactivity of the material to be tested in an organism with critical metabolic response. 5,6 The use of rats, mainly R. Norvegicus, Wistar rats, and Sprague-Dawley rats in these types of studies is consolidated. 7,8 In the case of in vivo experimental models with the properties of critical bone, the animal characterized by advanced age is the pioneer, receiving the name of senile. This animal is classified as being aged 15-18 months. 9 With aging, in addition to estrogen deficiency, molecular and cellular damage is accumulated and associated with the loss of specific tissue progenitor cells, generating a functional decline in tissue homeostasis, which manifests itself as osteopenia/ osteoporosis. Thus, bone physiology of senile rats is close to those found in women aged 48-55 years. 5,7,10 A well-established model for inducing osteoporosis found in the literature is ovariectomy (OVX model). 7 The rats are subjected to removal or ligation of the ovaries, and after 30 days, the depletion of estrogen levels is consolidated; 7 this implies an imbalance of bone turnover, in addition to reducing osteoblastic longevity, resulting in the prevalence of osteoclastic activity and bone resorption, leading to rapid loss of trabecular bone mass and osteopenia. 11,12 In bioactivity studies related to biomedical implants used to replace lost teeth or fixation of craniofacial or orthopedic fractures, different coatings methods have been shown to promote significant attraction of cells of the osteoblastic lineage and accelerate biological responses of mineralization bone. This is clinically justified in bones of low quality and density, such as conditions of osteoporosis, diabetes, irradiation, and osteonecrosis. Therefore, for these analyses, obtaining a more critical model allows the other biases of local or systemic compensations, that may interfere with the peri-implant bone tissue's biological response, to be eliminated.
The rat tibia for the bone repair assessment was used due to its resemblance to the microarchitecture of human alveolar bone, as reviewed by Glösel, et al. 13 (2010). It is even more similar to human alveolar bone than rat jaws in terms of bone density, besides being extensively used in the literature. [14][15][16][17][18][19] Thus, this study aimed to investigate the properties of critical bone in the main study models of osteoporotic animals found in the literature, comparing the animal model of senile rats and ovariectomized rats through the physiological responses obtained by cell culture (ex-in vivo) and installation of osseointegrated implants (in vivo).

Experimental groups
The 69 rats were randomly assigned to three experimental groups, which were further subdivided in ex-in vivo (n=9) and in vivo (n=60). These were the experimental groups:  Each group presented n=3 for the ex-in vivo study and n=20 for the in vivo study.
The experimental design is represented in a flow chart in Figure 1.

Animals Surgery
A total of 46 young adult rats (in vivo n=40 and ex-in vivo n=6) were randomly assigned to OVX group (bilateral ovariectomy) 20 and SHAM group (fictitious surgery). The protocol for the ovariectomy procedure was followed as described by Polo, et al. 14 (2020). Sodium Dipyrone 500 mg/kg and prophylactic antibiotic therapy with Pentabiotic 0.1 mg/kg of weight were administered in a single intramuscular dose each.
The animals from the three groups were euthanized (corresponding to the animals for the ex-in vivo stage) and the other animals were subjected to the implant installation (for the in vivo stage). For the serum dosage of estrogen (Estradiol -ng/mL), four animals from each group were sedated and euthanized by decapitation for blood collection, which was processed using the ELISA method. 14

Cell culture and isolation
After 90 days of the fictitious surgery or ovariectomy

Plating cells on titanium discs
The cells were trypsinized and plated on sterile Ti-6Al-4V alloy discs that were 10 mm in diameter and 2 mm thickness provided by Emfils Implantes Dentários

Cell Growth and Viability
For analysis at three, seven, and 10 days, cells were plated on four discs (2×10 4 cells per disc).
Through the assay of "3(4,5-dimethylthiazol-2-yl)-    according to the company's standards. Each animal received an implant in each tibial metaphysis. 14,16,17 For the placement of the implants, both tibial areas were incised until the periosteum exposed the metaphysis region. The cavitation was prepared using a 1.4-mm diameter drill in a handpiece operating at a speed of 800 rpm under abundant saline solution irrigation. The implants were installed using a manual key with 1.2 mm diameter.

Histological analysis
For the preparation of the slides, the rats were

Real-time PCR analysis
The real-time PCR analysis showed that gene expression was lowest in all analyzed proteins for the SENIL group when compared with the OVX group (p<0.05). When comparing the SENIL group with the SHAM group, there was a statistical difference only in the ALP protein (p>0.05); the other proteins showed similar results (Figure 3).

Alkaline phosphatase activity
In the analysis of alkaline phosphatase, the OVX group showed higher results than the SENIL group, with a statistical difference (p=0.001), and the SHAM group, with no significant difference (Figure 4).

Analysis of the formation of mineralized bone similar to nodules
The evaluation of the mineralized nodules confirmed that the SENIL group obtained less mineralization than the others. The OVX group also stood out in this analysis, obtaining a statistical difference when comparing the SENIL group with the OVX and OVX compared to the SHAM (p<0.001) ( Figure 5).

In vivo Histological parameters and biomechanical analysis (reverse torque)
In the qualitative histological analysis, both OVX and SHAM groups presented neoformation of bone tissue in the higher portions (peaks) of the bone/ implant interface, while in the lower regions (valleys), they presented a more significant amount of organized connective tissue. In the SENIL group, bone tissue neoformation occurred in the highest portion of the region corresponding to the implant thread, with a high amount of organized connective tissue. (Figure 6).

Biomechanical Analysis
In the biomechanical analysis between the groups, it was possible to verify more consistent data in the

Confocal laser microscopy (peri-implant bone dynamics)
In the analysis of fluorochromes by confocal microscopy, the SENIL group showed much higher values regarding the old bone (calcein/green) compared to the other experimental groups, presenting statistical differences from the SENIL group to the other groups. In the analysis of alizarin, the groups showed no statistical difference (Figure 7).

Histometry
As for the histometric analysis, the values assigned to BNF were higher for SHAM and OVX, with a statistically significant difference (p<0.05) compared to SENIL, which had the smallest area of BNF. In the analysis of the BIC, the values followed the same as the BNF, in which the SENIL group had the lowest values, with a statistically significant difference (p<0.05). The SHAM and OVX groups' values showed no statistical difference (p>0.05) (Figure 9).  analysis also corroborates the hypothesis mentioned above, since OVX animals obtained a greater number of mineralized nodules, followed by SHAM and finally by SENIL groups. 9,21,22 In the analyses related to reverse torque, histology, and confocal microscopy, the SHAM group showed a more significant amount of neoformed bone; as showed that OVX and SENIL groups presented similar values in many of the tests performed. A particularity is that the age considered senile was 12 months. 7,9,11 The histometric analysis is worth more attention